Utilizing TCR-Seq to Detect Tabelecleucel, an Allogeneic Epstein-Barr Virus (EBV)-Specific T-Cell Immunotherapy, Post-Infusion

Background: Tabelecleucel is an off-the-shelf, allogeneic EBV-specific T-cell immunotherapy that targets EBV-antigen presenting cells in an HLA restricted manner. It is not genetically modified and therefore lacks a distinct universal molecular moiety to detect it post-infusion. To date, the most effective measure of EBV reactive T-cells in patients (pts) treated with tabelecleucel is the EBV-cytotoxic T lymphocyte precursor (CTLp) assay, which measures the number of EBV reactive CTL precursors in each sample but cannot distinguish between the pt's endogenous CTLs or the infused product CTLs. We have previously shown that EBV-CTLp peak correlates with pt response. Prior methods to identify circulating product CTLs have relied on differences in the genomic profiles of the pt and dosing product lot. However, the limit of detection for these assays to date has not been sensitive enough to reliably detect product CTLs post-infusion. One aspect of T-cell biology that could be utilized to track product specific CTL clones is the unique T cell receptors (TCR) which impart antigen specificity. TCRs are highly diverse and only a small minority of TCR clones are found to be shared across unrelated individuals. Therefore, we leveraged TCRβ sequencing of the CDR3 region to barcode unique TCR clones in tabelecleucel product lots and pt derived samples to detect product T cell clones post-infusion, and annotate likely EBV-targeting T cell clones based upon sequence homology to previously reported EBV-targeting TCR clones. Using this approach, we aimed to identify corollaries associated with clinical outcomes.

Methods: Peripheral blood mononuclear cell samples from pts enrolled in the ALLELE (NCT03394365) study were obtained pre-dose on cycle (C) 1 day (D)1/D8/D15, C2D1/D8/D15 and at safety follow-up. EBV-CTLp was used to measure circulating EBV reactive T-cells, and both the pre-infusion baseline timepoint, and the timepoint of peak on-treatment EBV-CTL expansion were selected for downstream TCRβ sequencing. TCR repertoires for pt samples and the tabelecleucel dosing lot were assessed using TCRβ immunoSEQ assay. The captured TCR repertoires of pt samples were compared to the TCR repertoire of the dosing tabelecleucel lot to annotate product TCR clones and were compared against publicly available databases of previously identified EBV-specific TCRβ sequences to annotate putative EBV targeting TCR clones.

Results: Analyses for an initial subset of 7 pts with EBV-positive post-transplant lymphoproliferative disease have been completed and are described here. Six unique tabelecleucel lots were used to treat these pts and were included in the analysis. On average 0.15 ± 0.04% of TCRs detected were shared between multiple lots, allowing the ability to leverage TCRβ sequences as a unique barcode to identify specific product lots. All 7 pts tested had TCRβ clones matching previously identified EBV-specific TCRs detected at baseline (median [min, max]: 3498 [1855, 12486]) and peak EBV-CTLp; clones detected at peak EBV-CTLp were either those previously detected at baseline (532 [129,1627]) or newly emerged (3622, [1801,5347]). TCRβ clones matching the dosing lot were identified in 6 out of 7 pts. Three pts had TCRβ clones matching the dosing lot present at baseline prior to tabelecleucel infusion (2 [1,5]). Post-infusion emergence of TCRs matching the dosing lot was detected in 5 pts (1 [1,3]). TCRβ sequencing on additional pts is currently underway. Association of post-infusion detection of dosing lot specific TCR clones with pt best overall response is ongoing and will be reported at the time of presentation.

Conclusions: Utilizing TCRβ sequencing we can identify putative EBV-targeting TCR clones and dosing product TCR clones in pt TCR repertoires. Overall, an average of 12.5 ± 1.6% of captured TCR repertoires were annotated as EBV-targeting TCR clones. As publicly available reference datasets for EBV-specific TCRs are currently limited, this is likely an underrepresentation of the totality of EBV-specific TCRs present within the samples. Additionally, an average of 75.8% of the dosing product TCR clones detected were found exclusively after tabelecleucel infusion. Altogether, these results confirm that tracking TCRβ clones can be utilized as a novel assessment to detect post-infusion non-genetically modified allogenic cell therapies and future associations with clinical efficacy will be determined.

Ruiz:Atara Biotherapeutics: Current Employment, Current holder of stock options in a privately-held company. Spindler:Atara Biotherapeutics: Current Employment, Current holder of stock options in a privately-held company. Arthurs:Atara Biotherapeutics: Current Employment, Current holder of stock options in a privately-held company. Jehng:Atara Biotherapeutics: Current Employment, Current holder of stock options in a privately-held company. Widmann:Atara Biotherapeutics: Current Employment, Current holder of stock options in a privately-held company. Benoun:Atara Biotherapeutics: Current Employment, Current holder of stock options in a privately-held company. Ru:Atara Biotherapeutics: Current Employment, Current holder of stock options in a privately-held company. Mehta:Atara Biotherapeutics: Current Employment, Current holder of stock options in a privately-held company. Dinavahi:Atara Biotherapeutics: Current Employment, Current holder of stock options in a privately-held company. Dubovsky:Atara Biotherapeutics: Current holder of stock options in a privately-held company, Ended employment in the past 24 months. Nguyen:Atara Biotherapeutics: Current Employment, Current holder of stock options in a privately-held company.